Project Summary/Abstract The long-term goal of my research is to determine the mechanism by which the brain responds to the presence of nicotine and leads to voluntary regulation of its intake. The ultimate purpose is to develop a better understanding of how positively and negatively rewarding effects of this drug can lead to nicotine craving or nicotine aversive behavior, and how these two opposing effects are both regulated by the habenulo- interpeduncular circuitry. The studies detailed in this proposal will focus specifically on the neurophysiology of the medial habenula and the behaviors associated with activity of neurons in that region. Nicotine addiction, fed by regular tobacco smoking or chewing, or more recently by e-cigarette use, is a leading cause of death in both the developed and developing world. Nicotine acts in the body as an extremely potent agonist of the eponymous nicotinic acetylcholine receptor family (nAChR), which are ion channel proteins with functions in neurotransmission in the brain and at neuromuscular junctions in the periphery. NAChr are ubiquitous throughout the brain, and the mechanisms by which nicotine influences behavior to produce physiological dependency are complex. A specific nucleus in the epithalamus called the medial habenula has been implicated as a locus where circulating nicotine binds directly to a specific subtype of nAChR, in which genetic mutants have been found to be upregulated in heavy smokers, to produce downstream behavioral responses regulating voluntary nicotine intake. Intriguingly, a recently identified calcium-activated chloride channel called TMEM16A (Transmembrane protein of unknown function 16A) is very highly expressed in the medial habenula but almost nowhere else in the brain, and it is likely to contribute strongly to the firing properties of mHb neurons, though the mechanism by which nicotine produces its effects on the habenula and its associated circuitry is not known. I propose to investigate the mechanism of nicotine aversion in medial habenula neurons, and to begin by studying the TMEM16A channel as a functional contributor to nicotine aversion. In the first aim pursued during the K99 phase, I will use self-administration assays with direct habenular nicotine microinjections, as well as withdrawal assays with long term exposure followed by deprivation, to examine how acute and chronic nicotine affects behavioral responses mediated in the medial habenula and how knockout of the Tmem16a gene in mice affects those effects. In the second aim, I will begin to more generally probe the medial habenula's function by implanting microendoscopes and using in vivo fluorescence imaging experiments to directly visualize neuronal activity in response to acute and chronic nicotine exposure. In the third aim, taking place following the transition to independence, I will combine the in vivo imaging and self-administration paradigms to rigorously investigate medial habenular neuron function in regulating nicotine self-administration in wild-type mice and models with established nicotine- seeking phenotypes traceable to the medial habenula. I will also probe the cellular basis of plasticity during onset of nicotine dependence using RNAseq analysis of medial habenula neurons in nave and dependent mice. Finally, in aim 4, I will perform paired slice recordings of the medial habenula and the interpeduncular nucleus to investigate the input/output relationship of firing in the mHb, and how these are affected by focal applications of nicotine or other specific agonists, as well as by pre-exposure of the mice to chronic nicotine. Since joining the Jan lab in 2012, my research has focused specifically on the biophysics and pharmacology of the TMEM16A channel. Directly upon joining the lab, I undertook a short collaboration with Jan lab postdoc Fen Huang, Jason Rock, and others to explore the role of TMEM16A in airway mucus secretion and the ability of TMEM16A blockers to alleviate this in a cystic fibrosis model. Having a background in the structural biophysics of ion channel gating, I quickly became interested in how the TMEM16A channel is opened in response to elevation of intracellular calcium concentration (as would happen as a result of activation of a nicotine receptor, for instance), a study which resulted in a publication in the journal eLife, where I was co-first author. More recently, I performed a study to identify amino acids in TMEM16A important for chloride ion flux, and to characterize two novel inhibitory compounds with a high affinity for the TMEM16A ion pore. That study was published in PNAS in 2015. Over the remainder of my postdoctoral training, I would like to gain experience in experimental approaches to translate my expertise in the study of ion channel function into better understanding of how they contribute to the physiology of neuronal circuitry of the brain, with a specific focus on nicotine-sensitive circuitry involved in addiction and associated behavioral phenotypes. I believe the K99/R00 Pathway to Independence award is an ideal program for this goal, as it allows me to use both my specific areas of strength, and to acquire my desired training in the K99 phase to develop long term projects studying the neurophysiology of nicotine addiction and aversion in the R00 phase.